DNA Fragment Of Interest Research Articles

MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the European Union market

In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.

S55. ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia

Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive paralysis and ultimately respiratory failure within 5 years of symptom onset. Approximately 5–10% of ALS cases exhibit familial inheritance (FALS) and causative gene mutations can be found in 60% of FALS patients. The remaining 90–95% of ALS cases show sporadic manner (SALS) and mutations in the same genes are responsible for 10% of SALS patients. To date, rare variants in more than 30 genes have been reported to cause or be associated with ALS. Recently, Smith and et al. identified mutations in ANXA11 in ALS patients of European ancestry but pathogenicity of ANXA11 in other ALS cohorts remained unproved. In the current study, we investigated ANXA11 mutations in Chinese ALS patients with or without cognitive decline. Methods: Study population A total of 383 Chinese ALS patients were recruited at ALS clinic of Neurology Department, Peking Union Medical College Hospital (PUMCH) from January 2016 to August 2017. Mutation screening All coding exons and at least 100 bp of flanking introns of ANXA11(NM_145869) were sequenced using ABI 3730 automated DNA-sequencing system. Rare variants (MAF Transcripts investigation of the splicing mutation Total RNA was extracted using TRIzol (Invitrogen) from peripheral white blood cells. Then 2.5 μ g RNA were used to perform Reverse transcriptase-PCR. cDNA was amplified using primer pair ANXA11-RT-F(5′-CCATGAGCTACCCTGGCTAT-3′) and ANXA11-RT-R(5′-GACTCCCCAGGCAGTCAAT-3′) locating at ANXA11 exon 4 and exon 8 (shown in Fig. 3), respectively. The PCR products were isolated on a 1.5% agarose gel. DNA fragments of interest were gel-purified and sequenced. Results We identified 6 nonsynonymous heterozygous mutations (c.107C>G, p.P36R; c.119A>G, p.D40G; c.382G>A, p.V128M; c.687T>A, p.S229R; c.904C>T, p.R302C; c.1471G>A, p.G491R) and 1 splice-site mutation(c.174-2A>G, p.A58_Q187del) in 10 unrelated patients, accounting for a mutant frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS and 8.3% (1/12) in ALS-FTD. A novel p. P36R mutation was identified in one FALS index, one SALS patient and one ALS-FTD. The splicing mutation(c.174-2A>G) caused in-frame skipping of the entire exon 6 and further supported the loss-of-function mechanism. Conclusion ANXA11 gene is the most frequently mutated genes in Chinese patients with SALS. The study findings further expand the ANXA11 phenotype, firstly highlighting its pathogenic role in ALS-FTD.

The Association between Prothrombin Gene G20210A and Factor V Leiden Mutation in Women with Complications of Pregnancy in Baghdad Province

Factor V Leiden (FVL) (G1691A) and prothrombin gene (G20210A) mutations are the most common inherited forms of thrombophilia. The main objective of this study is to analyze the association between inherited thrombophilia FVL mutation and prothrombin G20210A mutation and recurrent pregnancy loss (RPL) among women suffering from complications of pregnancy. The study included 40 buccal swab samples collected from women at the reproductive age complications; abruption placenta 12.5%, dead fetal 37.5%, and recurrent spontaneous abortions (RSA) 50% in comparison with 30 women who had one or more normal pregnancies from Elwiyah Obstetric teaching hospital through the period from mid of September 2014 to the mid of March 2015. The median age of patients was 32 years (range: 19–42 years) while for the control group, it was 28 years (range: 17–41 years). Out of 40 women, 65% had one pregnancy loss and 35% for more than two previous pregnancy losses. According to the time of pregnancy losses, 22(55%) women had early pregnancy loss (EPL), and 18 (45%) women had Late Pregnancy Loss (LPL). For FVL mutation detection the restriction fragment length polymorphism (RFLP) was used. DNA fragment of interest was amplified by PCR and then subjected to digestion by MnlI specific restriction enzyme. For prothrombin G20210A mutation detection different PCR products were generated using a set of primers in A/A, G/G, A/G alleles. Out of the 70 samples tested for FVL mutation no homozygous FVL mutation was detected and Prothrombin gene mutation G20210A was totally absent among patient and control.

Isolation and molecular characterisation of actin gene of Trypanosoma evansi from Indian dromedaries

The present study was carried out to isolate the actin gene of Trypanosoma evansi by reverse transcription polymerase chain reaction, clone the amplicon in a suitable bacterial plasmid vector, characterisation of gene through sequencing and molecular modeling of obtained actin protein. Total RNA of T. evansi was used to amplify the actin gene by RT-PCR, amplification was done using gene specific primers and desired amplicon was identified on the basis of size of the gene. For cloning, DNA fragment of interest was then ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. After cloning, screening of recombinants was done by restriction enzyme digestion of plasmid DNA. After confirmation of clone, the plasmid DNA was sequenced and coding sequences of actin gene was of 1131bp. Sequence analysis revealed that actin gene of Trypanosoma evansi from Indian isolate shared 81.4–99.5% and 93.4–98.7% sequence identity at the nucleotide and amino acid level, respectively with isolates from different geographical areas of the world.

Isolation, PCR Amplification and Cloning of Heat Shock Protein Gene from Salivary Glands ofHyalomma dromedariiTicks fromCamelus dromedarius

A molecular study was carried out to isolate the heat shock protein gene of Hyalomma dromedarii ticks of camels, which play important role in their survival under harsh environmental conditions. Engorged adult H. dromedarii ticks were collected from camel herds in the Bikaner district of Rajasthan. Total genomic DNA and RNA was isolated from the salivary glands of ticks. Primers were designed for amplification of heat shock protein gene from H. dromedarii by using base sequence of Haemaphysalis longicornis. The heat shock protein gene of H. dromedarii was successfully amplified from genomic DNA and cDNA and was identified on the basis of its size in agarose gel electrophoresis as 560 bp. The amplicon of expected size was purified from the 1% low melting agarose gel. DNA fragment of interest was then ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains for cloning. Screening of recombinants was done by restriction enzyme digestion of plasmid DNA and by colony PCR for quick screening of plasmid insert directly from E. coli colonies in the presence of insert specific primers.

An organic acid based counter selection system for cyanobacteria.

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).

Ser49Gly polymorphism in the β-adrenergic receptor 1 gene in a population sample from Rio de Janeiro state, Brazil, stratified by self-identified skin color and genetic ancestry.

Cardiovascular diseases (CVD) have the highest worldwide mortality rate of any type of disease. In recent years, genetic research regarding CVD has been conducted using association studies, in which the presence of a genetic polymorphism associated with a specific cell signaling pathway in a lower or in a higher frequency among patients may be interpreted as a possible causal factor. Genetic polymorphisms that occur in the β-adrenergic receptor 1 (β-ADR1) can result in significant changes in its function that may result in physiopathologies. Ambiguous categorizations, such as skin color and self-reported ethnicity have been used in pharmacogenetic studies as phenotypic proxies for ancestry; however, admixed populations present a particular challenge to the effectiveness of this approach. The main objective of the present study was to estimate the diversity and the frequency of the Ser49Gly polymorphism of the β-ADR1 gene in a sample of 188 male individuals from the population of Rio de Janeiro. The Ser49Gly frequencies were analyzed by two forms of sample stratification: The phenotypic criterion of black or non-black skin color, and African or non-African ancestry, defined using Y-chromosome single nucleotide polymorphisms and autosomal indel markers. These results were used to evaluate whether marker-based ancestry criteria and/or skin color were associated with the frequency of the Ser49Gly polymorphisms in the heterogeneous Rio de Janeiro/Brazilian population. The DNA fragments of interest were amplified by polymerase chain reaction with specific primers for the Ser49Gly marker, and genotyping reactions were performed by restriction with the enzyme Eco0109I. Heterozygosity values ranging from 0.25 to 0.50 and 0.20 to 0.41 were found for the groups stratified by ancestry and skin color, respectively. Using the Hardy-Weinberg equilibrium at the ser49Gly marker, it was found that there was no significant deviation in the genotype distribution of the whole Rio de Janeiro sample or the stratified sample. Analysis of the allelic distribution in the Rio de Janeiro population sample revealed frequencies of 80.30 and 19.70% for the wild-type (Ser49) and mutated (Gly49) alleles, respectively. Significant differences were observed in the allele frequencies of the Ser49Gly marker between the self-defined black and non-black phenotype, and the African and non-African descendant genotype population samples. A significant difference was also observed between blacks and African-descendant individuals, with a lesser degree of genetic differentiation. The results presented in the present study suggest that the Ser49Gly marker has a distribution that is influenced by an ancestral component, due to the increased prevalence of the Gly49 polymorphism in the black and African descendant populations of the Rio de Janeiro state. This evidence, in combination with clinical studies, may contribute to a detailed analysis of the pattern of susceptibility to CVD involved in β-ADR1 receptor mechanism failure.

A dual color fluorescent reporter system for the real time detection of promoter activity

Understanding the mechanisms controlling transcription of a gene requires the identification and characterization of its cis-acting regulatory elements. A highly useful approach to the identification and characterization of cis-acting elements has been the systematic coupling of genomic fragments to reporter constructs, so called "promoter bashing". The expression from such reporters must be normalized for differences in transient transfection efficiency between cells and replicates. A novel dual color fluorescent reporter system to assay the promoter activity of a genomic DNA fragment of interest was established by cloning a Discosoma red fluorescent protein gene and a green fluorescent protein gene into a single vector, giving a system in which the ratio between red and green fluorescence is proportional to promoter activity. This system allows real time quantitative monitoring of promoter activity. We validated this approach by assaying the cis-acting regulatory potential of the peroxisome proliferators-activated receptor gamma2 gene.

Micrococcal Nuclease-Southern Blot Assay: II. Capillary Transfer and Hybridization: Figure 1.

INTRODUCTIONThis is the second phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. In this second part of the assay, the restricted genomic DNA is separated by agarose gel electrophoresis and stained with ethidium bromide, resulting in a ladder of bands corresponding in size to multiples of the nucleosome core plus linker (~200 bp). To determine whether a DNA fragment of interest is nucleosomal, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes.

Micrococcal Nuclease-Southern Blot Assay: I. MNase and Restriction Digestions: Figure 1.

INTRODUCTIONThis is the first phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. Micrococcal nuclease (MNase) is unique among nucleases in its relative ability to induce double-strand breaks within nucleosomal linker regions, but only single-stranded nicks within the nucleosome itself. Because of this property, micrococcal nuclease can be used to determine whether a DNA fragment of interest is nucleosomal. In this phase of the assay, the cells are lysed and nuclei are isolated. Limiting concentrations of micrococcal nuclease are added to the nuclei, resulting in cleavage at nucleosome linker regions (preferably cleavage at two sites per DNA molecule). The cleavage reactions are stopped, and then the genomic DNA is purified and digested with restriction enzymes.

Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro

Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to <5 x 10(2) copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.

Manipulating biomolecules in space and time

Complex biological systems demand high-throughput, costeffective, high-sensitivity analysis techniques. For example, the determination of gene function, or genomics, promises to help unravel and treat many genetic disorders, such as cancer. For the past decade, researchers have monitored gene function using microarrays, in which various DNA fragments or proteins are arranged on a plate to simultaneously detect thousands of different biomolecules extracted from cells. To allow genomic analysis within living cells, microarrays of cells have also been investigated. Of particular interest are transfected cell microarrays (TCMs),1, 2 first developed by Ziauddin and co workers.3 Here, an array of cell clusters is formed, such that each is transformedwith a different DNA sequence. In brief, spots of various DNA fragments of interest are first arrayed on a glass slide at addressable locations. Cells are then seeded over the array, and those that attach on top of particular DNA spots take up and express that DNA, a process termed ‘solid-phase transfection.’ Creating these devices requires surfaces that control the behavior of cells and of the biomolecules they use to interact with surfaces, so that adjacent DNA spots and cell clusters are effectively separated to prevent cross-talk. Furthermore, efficiently transporting the DNA sequences of interest to, and inserting them inside, the cells remains a challenge. For this purpose we have developed a surface that is able tomanipulate biomolecules in both space and time, for application as a TCM.4 Our process is illustrated in Figure 1. The surface is constructed by first depositing an allylamine plasma polymer (ALAPP) that contains amine functionality. An aldehydefunctionalized poly(ethylene glycol) (PEG) chain is then grafted onto the surface by reductive amination. Finally, laser ablation of this surface through a mask removes the PEG layer— Figure 1. Schematic of the formation of a transfected-cell microarray (TCM). A boron-doped, p++ silicon substrate was modified sequentially by plasma polymerization and poly-(ethylene glycol) (PEG) grafting. Laser ablation was used to expose allylamine-plasma-polymer (ALAPP) wells within the PEG surface. A robot was then used to spot DNA into these wells. Cells seeded onto this surface grew exclusively within the wells, over the DNA spots. Application of a negative voltage triggered the release of DNA from the surface, making it available for uptake by adhered cells. Transfected cells are depicted as green. The schematic is not drawn to scale. Reprinted from Reference 4.

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Analysis of site-directed mutagenesis constructs by capillary electrophoresis using linear polymer sieving matrices

Site-directed mutagenesis is a novel molecular biology tool, which introduces mutations into DNA fragments of interest in a well-defined manner. Sequences with designed mutations can be generated in this way to express altered protein sequences for structure–function relationship studies. However, prior to gene expression, it is important to analyze the DNA construct to see whether the introduction of the mutation was indeed successful. Currently DNA sequencing is the method of choice for this verification. This paper introduces the combination of primer extension and capillary electrophoresis using linear polymer sieving matrices as an efficient alternative for this type of mutation analysis. The site-directed mutagenesis construct served as template in the primer extension reaction that employed a fluorophore labeled primer in close proximity to the mutation. Appropriate ddNTP was used to block the extension when the mutation was present, while the other three dNTPs enabled elongation of the primer. Alternatively, non-labeled primers can be used with the proper fluorophore labeled ddNTPs to block the reaction. Rapid analysis of the labeled primer extension products (mutant or wild type) was obtained by capillary electrophoresis using denaturing sieving matrix and laser-induced fluorescence detection.

Pellet Pestle Homogenization of Agarose Gel Slices at 45°C for Deoxyribonucleic Acid Extraction

A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris–EDTA buffer), containing the DNA fragment of interest, at 45°C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The “homogenate” is then centrifuged for 30 s and the supernatant is saved. The “homogenized” agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70–90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.

Random mutagenesis by using mixtures of dNTP and dITP in PCR.

Generating random mutations in a DNA fragment of a specific size is often the method of choice for changing specific properties of an encoded protein or for studying the fimctional properties of a specific DNA fragment. In all these cases it is helpful and in some cases essential to have a screening method available that allows for distinction between phenotypic changes brought about by one or more random mutations in the DNA fragment of interest. Several methods have been described using mixed synthetic oligonucleotides for random mutagenesis, which work efficiently but can only be applied for mutagenesis of DNA fragments of limited size (1—3). Alternative methods using polymerase chain reaction (PCR) with reduced fidelity have been described that offer an increased mutation frequency as well as the advantage of random mutations in relatively large DNA fragments and a random distribution of the types of mutation (4—6). However, for many applications it is desirable to use a method that allows the modulation of the desired number of mutations in a DNA fragment of a specific size and the degree of randomness of the mutations. The method described here (7) offers the advantage of choosing preferred nucleotides being randomly incorporated as well as adjusting the mutation frequency to almost any desired level. This method is based on the following principles:

Self-cloning in filamentous fungi: application to the construction of endothiapepsin overproducers in Cryphonectria parasitica

The filamentous ascomycete fungus Cryphonectria parasitica naturally secretes endothiapepsin, an aspartic proteinase. It is cultured on a commercial scale as a source of the milk-clotting enzyme for cheese making. Our objective was to increase enzyme production of an industrial C. parasitica strain by a new technique of self-cloning; it consisted in the screening for transformants producing higher levels of endothiapepsin and having integrated only the DNA fragment of interest. Such genetically improved strains that are devoid of any foreign genes should be more readily acceptable for the production of food-grade enzymes.

A simple method for randomly mutating cloned DNA fragments by using chemical mutagens and the polymerase chain reaction

Random mutagenesis of cloned DNA fragments can be an important tool for understanding genes and/or gene products. I report here a simple procedure for generating random mutations in any cloned DNA fragment in vitro. This method utilizes the polymerase chain reaction (PCR) to amplify chemically damaged single-stranded DNA containing the DNA fragment of interest. A mutagenized pool of plasmid DNA is produced by cloning the PCR product (containing chemically induced sequence heterogeneity) into a new vector. Therefore, extreme chemical treatments can be used that biologically inactivate the original vector. In this report, 10% of the plasmids contained a mutation in a 97-nucleotide DNA insert, giving a mutagenesis frequency of 10 −3/base pair. Of 3000 yeast transformants screened, seven intron mutations were isolated that activate a cryptic 3′ splice site, suggesting that 2% of the mutations could give rise to the desired phenotype. The seven mutations included transitions, transversions and single nucleotide deletions, demonstrating the well-balanced repertoire of nucleotide changes derived from this procedure.

New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage λ attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the λ attachment site attP and (ii) a helper plasmid, bearing the λ int gene, transcribed from the λ P R promoter under the control of the temperature-sensitive repressor cI 857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 °C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 °C. These vectors have been successfully used for integration of several promoter- lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.

Diagnosis of tuberculosis by polymerase chain reaction

For the diagnosis of extrapulmonary tuberculosis in adults and all forms of tubercular infections in children, microscopic and cultural techniques have been shown to be inadequate. Many serological techniques have been employed for non culture diagnosis of tuberculosis. Early promising results have repeatedly given way to subsequent findings of non-specificity. Major mycobacterial antigens have been shown to be heat shock proteins which are highly conserved in nature. DNA probes for tuberculosis are specific but have a sensitivity equivalent to AFB smear examination. Polymerase Chain Reaction (PCR) with its ability to selectively amplify DNA fragments of interest offers a potentially powerful technique for the rapid, specific and sensitive diagnosis of tuberculosis. Samples from partially treated patients could be culture negative but can be detected by PCR.